Exploitation of the necrosis avidity of hypericin

Hypericin, a naturally occurring red fluorescent photosensitizer, has been of interest in severalresearch domains over the last years. Even though it was initially investigated as an antiviral agent,especially the tumoritropic properties of hypericin have shown to be of great potential in diagnosis and photodynamic treatment of superficial tumors such as bladder cancer. More recently it was discovered that hypericin robustly accumulates in necrotic tissue. Considering the abundant presence of necrotic areas in fast-growing solid tumors, possible diagnostic and therapeutic applications for hypericin in an oncologic setting were investigated. In the first part of our research project, imaging of necrotic tumors was evaluated using hypericin and radiolabeled monoiodohypericin. By means of fluorescence microscopy, autoradiographic imaging and planar gamma scintigraphy, it was possible to visualize necrotic tumor areas on microscopic and macroscopic scale. Results showed that hypericin displays a high retention in necrotic compared to viable tissue with optimal contrast observed starting from 24 h after administration. Furthermore, an explorative tumor necrosis therapy (TNT) study was carried out using 131I-HYP in order to investigate a possible therapeutic application of the necrosis avidity of hypericin. A mild antitumor effect was observed in moderately sized tumor that developed spontaneous necrosis only after the third administration, when tumor size had grown past 2 cm3. Despite the need for optimization of this therapeutic concept, a proof of principle had been established for TNT using a small molecule. Indeed, a following therapy study using a vascular damaging agent (VDA) to induce larger areas of necrosis showed a markedly better result compared to targeting spontaneous necrosis. In order to further optimize the concept of this dual targeting strategy, the accumulation of hypericin in VDA-treated necrotic tumors was evaluated. Results showed a typical distribution of hypericin that concentrated at the periphery of large necrotic areas. Moreover, accumulation did not change when VDAs with a different mechanism of action were used. A decrease in accumulation could be observed, however, when hypericin was administered 1 h before administration of the VDA. Lastly, we noticed cell-shaped uptake of hypericin in nontarget tissue, which is likely attributed to phagocytic clearance from necrotic areas. Moreover, this cellular clearance was observed less when a tubulin-binding VDA was used compared to a cytokine-inducing VDA. Immunosuppression using dexamethasone did not alter this effect, nor did it influence uptake of hypericin in necrosis. Because iodination is the most convenient way to radiolabeling of hypericin without greatly altering its molecular structure and physicochemical properties, 123I-HYP and 131I-HYP were preferentially used in the past for TNT and radio-imaging.However, iodine has some drawbacks: upon deiodination, isotopes may accumulatein nontarget organs such as the thyroid gland. Moreover, isotopes such as 90Y and 99mTc exist, with favorable characteristics when used in imaging and radiotherapy, respectively. To incorporate other radioisotopes in the targeting of necrotic tissue with hypericin, a pretargetingstrategy was devised, physically separating the effector isotope from hypericin. Since this can beachieved by making use of the strong interaction between biotin and avidin, a conjugate of hypericin and biotin was synthetized. This biotinylated hypericin showed to be more hydrophilic than hypericin, yet less than hypericin monocarboxylic acid as determined by reversed-phase liquid chromatography. The conjugate was then evaluated in a two-step pretargeting strategy, consisting of two subsequentadministrations: first hypericin-biotin was allowed to accumulate into necrotic tissue. After this,radiolabeled avidin was injected, in turn accumulating in areas with high hypericin uptake. Indeed,autoradiographic analysis of necrotic tumor sections revealed that accumulation of avidin in necrotic tissue was higher when hypericin-biotin had been administered before. Conversely, it was also observed that avidin was not present homogenously throughout necrotic areas, indicating that this protein may not diffuse as efficiently as hypericin-biotin in this microenvironment. Furthermore, biodistribution of radiolabeled avidin revealed that there was a low absolute retention of avidin in necrotic tumor tissue. Comparably low retention was found when injecting radiolabeled avidin 24 h after hypericin-biotin and biotin-fluorescein were administered directly into the necrotic tumor. Despite showing the feasibility of pretargeting using a small molecule such as biotinylated hypericin, we concluded that this strategy would need further polishing in order to achieve retention comparable to straightforward administration of hypericin. In the last part of this research project, we searched for the mechanism underlying the necrosis avidity of hypericin. In an ex vivo approach, we performed subcellular centrifugation of necrotic liver tissue containing hypericin in order to investigate in which fraction accumulation was highest. Interestingly, most fluorescence was observed in the 600 G pellet, which contains the largest and heaviest cellular components. Moreover, we found that hypericin localization in necrotic areas in vivo and ex vivo co-localized with the fluorescent polyene antibiotic filipin, which binds to cholesterol. These observations suggest that there is an abundance of cholesterol-rich structures in necrotic tissue, possibly capturing hypericin resulting in its necrosis avidity. The results of this research provided supportive data enabling further development of TNT using the unique advantagesof VDA and 131I-HYP in a combination therapy. Furthermore, an alternative method towards the approach of TNT was explored using a pretargeting strategy that could allow the use of different radionuclides upon further optimization. Lastly, we investigated the possible mechanism underlying the necrosis avidity of hypericin. Despite the fact that no specific target was identified, further analysis and comparison of results obtained with hypericin to similar analysis of other NACAs, may reveal a common mechanism of action, stimulating the discovery and development of novel NACAs.

Publication year:2012

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